Recent reports indicate that human immune deficiency virus (HIV) infects macrophages and monocytes in addition to T cells. Levy et al. (1985) Virology, 147:441-448; Gartner et al. (1986) Science, 233:215-219; and Wiley et al., Proc. Natl. Acad. Sci. USA, 83:7089-7093 (1986). Acquired immune deficiency syndrome (AIDS) results from infection with HIV, also known as human T-lymphotropic virus Type III (HTLVIII).
AIDS is characterized by extensive immunosuppression that predisposes patients to life-threatening opportunistic infections as well as unusual forms of neoplasm. As to the other known subgroups or types of human T-lymphotropic viruses, Type I (HTLV-I) is believed to cause T cell proliferation in leukemia. The role of HTLV-II in pathogenesis remains unclear, although it has been associated with rare cases of the T cell variant of hairy cell leukemia. Golde et al. (1986), Seminars in Hematol. 23:3-9.
Synthesis of DNA complementary to viral RNA is thought to be required for both retroviral integration into host DNA and for the generation of new virions. For this reason, the HIV-encoded reverse transcriptase is a logical target for the development of agents for the treatment of patients with the acquired immunodeficiency syndrome (De Clercq et al., J. Med. Chem., 29:1561-1569 (1986)], and with other diseases of retroviral origin.
Mitsuya et al. (1985) Proc. Natl. Acad. Sci. USA, 82:7006-7100 reported 3'-azido-3'-deoxythymidine (AZT) blocked the replication of HIV in cultured human T lymphoblasts, and inhibited the cytopathic effects of the virus. AZT was presumably phosphorylated by the T cells and converted to the 5'-triphosphate derivative. That derivative was reported by those authors to be an inhibitor of HIV reverse transcriptase activity. Yarchoan et al. (1986) Lancet, 1:575-580, administered AZT to patients with AIDS or AIDS-related disease complexes. The drug was reportedly well tolerated and crossed the blood/brain barrier.
Recently, Mitsuya et al. (1986) Proc. Natl. Acad. Sci USA, 83:1911-1915 reported that the 2',3'-dideoxynucleoside derivatives of adenosine, guanosine, inosine, cytidine and thymidine also inhibited the infectivity and cytopathic effect of HIV in vitro at concentrations from 10-20 fold less than those that blocked the proliferation of uninfected T cells. These compounds were also reported to be relatively non-toxic towards host T cells. The adenosine and cytidine derivatives were reported to be more potent than the guanosine and inosine derivatives.
The 2',3'-dideoxynucleosides are phosphorylated at the 5'-position in T cells to form the 5'-nucleotide triphosphate derivatives. Those derivatives are well known to be substrates for reverse transcriptase molecules. Ono et al. (1986) Biochem. Biophys. Res. Comm., 2:498-507.
Those 2',3'-dideoxynucleoside 5'-triphosphates are also utilized by mammalian DNA polymerases beta and gamma. Waquar et al. (1984) J. Cell. Physiol., 121:402-408. They are, however, poor substrates for DNA polymerase-alpha, the main enzyme responsible for both repair and replicative DNA synthesis in human lymphocytes. In part, these properties may explain the selective anti-HIV activity of the 2',3'-dideoxynucleosides.
Chan et al. (1982) J. Cell Physiol., 111:28-32 studied the pathways of pyrimidine nucleotide metabolism in murine peritoneal macrophages and monocytes, and reported undetectable levels of deoxycytidine kinase or thymidine kinase in these cells. High levels of adenosine kinase were found, however.
Similar high levels of adenosine kinase have been found in human monocytes and human monocyte-derived macrophages (MDM) in work carried out in the inventors' laboratory. In that preliminary work, MDM were found to exhibit about one-tenth to about one-fourth the nucleoside kinase activity of GEM T lymphoblasts (e.g. ATCC CCL 119) toward uridine, deoxycytidine and thymidine, and about two-thirds the adenosine kinase activity of GEM cells. In addition, that adenosine kinase activity of MDM cells was at least about 10-fold higher than any of the other kinase activities. Those studies also indicated relatively low levels of nucleoside phosphorylation using AZT, dideoxycytidine (ddC) and 2',3'-dideoxyadenosine (ddA) in intact GEM T lymphoblasts and still lower levels with the MDM.
The ability of AZT, ddC and ddA to inhibit synthesis of the p24 (gag) antigen of HIV in GEM and MDM cells was also examined. For GEM cells, the results for all three compounds were similar to those discussed in Mitsuya et al. (1987) Nature, 325:773-778 with ddC providing the most inhibitory effect at the lowest concentration, followed by AZT, followed by ddA in a 3-day assay. Using the same concentrations (0.1-100 .mu.M) in a similar 3-day assay, none of those compounds provided any inhibition of p24 (gag) production from MDM cells.
The above results explain in part the observations made in clinical trials with AZT. Those results, in part, have shown that treatment of patients with AIDS or AIDS-related complex with AZT has resulted in elevation of CD4 (T4) peripheral blood cell counts, restoration of cutaneous delayed hypersensitivity, and reduction of the rate of opportunistic infections and death; results that can be related to the effect of AZT on T cells.
However, AZT had no effect on virus isolation rates from peripheral blood cells. That result suggests that a subset of infected cells persists that represents a reservoir of continuing viral replication, and with the above work with MDM cells, indicates that macrophages constitute at least a portion of that in vivo reservoir of HIV.
As noted before, 2',3'-dideoxyadenosine (ddA) inhibits in vitro infectivity and cytopathic effects of HIV. Mitsuya et al. (1986) Proc. Natl. Acad. Sci. USA, 83:1911-1915. However, ddA is a known substrate for adenosine deaminase (also known as adenosine aminohydrolase, EC 3.5.4.4), which converts the compound to 2',3'-dideoxyinosine (ddI). Frederiksen (1966) Arch. Biochem. Biophys., 113:383-388. Adenosine deaminase levels in the blood cells of AIDS patients are relatively high compared to normal persons. Thus, in vivo, ddA would be expected to be degraded rapidly to 2',3'-dideoxyinosine, due to the action of endogenous adenosine deaminase. Although 2',3'-dideoxyinosine has anti-HIV activity, it is less potent than AZT or ddA (Mitsuya, 1986, above).
Several 2-substituted adenosine derivatives have been reported not to be deaminated by adenosine deaminase. For example, Coddington (1965) Biochim. Biophys. Acta, 99:442-451 reported that deoxyadenosine-1-N-oxide, as well as 2-hydroxy-, 2-methyl-, 2-chloro-, 2-acetamido-, and 2-methylthio-adenosines were neither substrates nor inhibitors for adenosine deaminase. Montgomery, in Nucleosides, Nucleotides, and Their Biological Applications, Rideout et al. eds., Academic Press, New York, page 19 (1983) provides a table of comparative K.sub.m and V.sub.max data for the deamination of adenosine, 2-halo-adenosines 2-halo-deoxyadenosines and 2-fluoro-arabinoadenosine that also indicates that those 2-halo adenine derivatives are poor substrates for the enzyme relative to adenine itself. Stoeckler et al. (1982) Biochem. Pharm., 31:1723-1728 reported that the 2'-deoxy-2'-azidoribosyl and 2'-deoxy-2'-azidoarabinosyl-adenine derivatives were substrates for human erythrocytic adenosine deaminase, whereas work of others indicated 2-fluoroadenosine to have negligible activity with adenosine deaminase.
2-Chloro-2'-deoxyadenosine is phosphorylated by non-dividing (normal) human peripheral blood lymphocytes and is converted to the 5'-triphosphate. This adenine derivative is not catabolized significantly by intact human cells or cell extracts, and is phosphorylated efficiently by T lymphocytes. Carson et al. (1980) Proc. Natl. Acad. Sci. USA, 77:6865-6869.
As discussed before, high levels of adenosine kinase have been found in murine peritoneal macrophages and in human monocytes. Adenosine kinase can phosphorylate 2'-deoxyadenosine derivatives, but does so less efficiently than deoxycytidine kinase. Hershfield et al. (1982) J. Biol. Chem., 257:6380-6386.
In addition to AIDS, other infectious diseases in which pathogenic organisms persist in chronically infected monocytes/macrophages are Chagas' disease and other trypanosomal diseases, Leishmaniasis, mycobacterial infections, systemic and local fungal diseases, and protozoal infections such as toxoplasmosis, malaria and pneumocystis.
Similarly, many autoimmune diseases share common features with the pathogenesis of viral infection. The specific mechanism that mediates autoimmune disorders can be augmented by amplification systems which may involve lymphokines or humoral components.
One form of autoimmune disease involves a cytotoxic mechanism wherein circulating autoantibody reacts with self-antigen present on a cell surface. The cytotoxic process can be mediated by complement or by cells as in antibody-dependent cell-mediated cytotoxicity. The end-result of the cytotoxic mechanism is usually cell lysis, elimination or inactivation, and this is the mechanism of many autoimmune hematologic disorders.
A second form of autoimmune disease involves the formation of immune complexes of autoantibody plus self-antigen that can fix complement as well as initiate inflammatory processes. Organs in which such complexes deposit are subject to inflammation, and ultimately to destruction. Nucleic acids are known to serve as antigens for this mechanism in systemic lupus erythematosus (SLE). Immune complex deposition appears to account for the glomerulonephritis present in many autoimmune disorders.
A third mechanism for autoimmune disorders is mediated by interactions of cells or their soluble products with antigen (cell-mediated immune response) rather than with antibody and complement (humoral immune response). This mechanism is classically manifested in delayed hypersensitivity, which is characterized by a reaction that is time-dependent, has a specific histologic sequence in terms of inflammation and cellular infiltration, and can only be transferred by cells and not by serum.
The effector mechanism of cell-mediated cytotoxicity can include direct cell interaction with antigen or elaboration of lymphokines and monokines. The lymphokines primarily amplify the initial reaction by nonspecifically recruiting inflammatory cells such as neutrophils, cytotoxic T cells, monocytes and macrophages to the reaction area. At that inflammatory site, a cascade effect occurs wherein cells become activated, proliferate and secrete more cytokines.
Rheumatoid arthritis is a chronic recurrent systemic inflammatory autoimmune disease primarily involving the joints. Recent studies have suggested that a virus, possibly Epstein-Barr virus, may be implicated in this autoimmune disorder. The Epstein-Barr virus is a polyclonal stimulator of B cells and can stimulate the production of rheumatoid factors by B cells. In rheumatoid arthritis, there is an increase in alpha.sub.2 -globulin, a polyclonal hypergammaglobulinemia, and hypoalbuminemia. Cryoprecipitates of immunoglobulins are often seen in rheumatoid vasculitis.
Rheumatoid factors can be present in other autoimmune disorders, as well as in rheumatoid arthritis. Rheumatoid factors have been found to be present in some patients with systemic lupus erythematosus, Sogren's syndrome, scleroderma and polymyositis.
The deposition of immune complexes on or in the synovia of joints appears to initiate or exacerbate an inflammatory response of the synovial membrane in rheumatoid arthritis. The deposited complexes fix and activate complement, which subsequently stimulates the attraction of inflammatory cells such as monocytes and macrophages. The deeper layers of the synovium are infiltrated by both T and B lymphocytes, plasma cells, monocytes, macrophages and occasionally neutrophils. The infiltrating cells elaborate several effector molecules of the inflammatory response, which transforms the joint fluid into an inflammatory exudate. The immune complexes together with the infiltrating cell-released factors activate the clotting pathway leading to fibrin production and deposition in the joint space, synovium and cartilage.
The essential role of the monocyte in rheumatoid arthritis was confirmed by Fujii et al. (1990) Ann. Rheum. Dis., 49:497-503. Peripheral blood and synovial fluid were obtained from 44 patients with rheumatoid arthritis. The obtained monocytes were examined for their ability to produce interleukin-1.beta., leucotriene B.sub.4, and prostoglandin E.sub.2, all factors important in the establishment and maintenance of chronic inflammation. Monocytes derived from the rheumatoid arthritis patients produced significantly more of these factors than monocytes obtained from normal patients, indicating that the monocytes in individuals with rheumatoid arthritis play an important role in mediating the chronic inflammation characteristic of the disease.
Multiple sclerosis (MS) is another monocyte-mediated chronic inflammatory autoimmune disease. Pathologically, MS is the result of demyelination in the brain and spinal cord (central nervous system). Symptoms resulting from this demyelination include weakness, visual impairment, incoordination, and paresthesia (abnormal tingling). The course of the disease is largely unpredictable, but often progresses through a cycle of exacerbation of symptoms followed by remission.
To date, usual treatment consists of therapy with ACTH or corticosteroids such as prednisone. Controlled studies suggest that such treatments induce more rapid clearing of acute symptoms and signs but leave the long-term outcome of the disease unaffected. Long-term maintenance therapy with ACTH or corticosteroids is contraindicated. Evidence indicates that immunosuppreessant agents have no long-term benefit. Cecil, Textbook of Medicine, Beeson et al., eds., 15th ed., W. B. Saunders Company, Philadelphia, (1979) page 847.
Although the exact etiology of MS is unknown, its origins are thought to be autoimmunologic. For example, experimental allergic encephalomyelitis (EAE), an animal model of demyelinating diseases such as MS, is induced by immunizing mice with whole myelin or specific myelin components such as myelin basic protein.
In humans with MS, exacerbations are correlated with high levels of neopterin in blood and cerebrospinal fluid. Neopterin is a factor released from activated monocytes and macrophages, thereby implicating these cells as being involved in MS exacerbations. Fredrickson et al., Acta Neurol. Scand., 75:352-355 (1987); Huber et al., J. Exp. Med., 160:310-316 (1984). Elevated neopterin levels are also found in patients with rheumatoid arthritis.
Indeed, at the microscopic level, monocytes and microglial cells (macrophages of the central nervous system) are found within the demyelinated regions of the nerve cells during MS exacerbations. Beeson et al. (eds.) Cecil Textbook of Medicine, W. B. Saunders Co., Philadelphia, Pa. (1979). As in rheumatoid arthritis, the monocyte plays an important role in mediating the inflammation responsible for MS.
Various usual treatment methodologies have been employed to ameliorate the symptoms of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis. Many of these are directed to use of palliative, anti-inflammatory agents.
Nonsteroidal anti-inflammatory agents, such as phenylbutazone, indomethacin, fenoprofen, ibuprofen, naproxen, sulindac, tolmetin, methotrexate, and mefenamic acid, and antimalarial drugs, such as chloroquine and hydroxychloroquine, have been employed for arthritis, but possess serious side effects upon prolonged usage. Salicylates are commonly employed, specifically aspirin, in dosages from about 3.6 to about 5.4 grams (g) per day. Numerous side-effects are associated with high-dose aspirin therapy such as gastric upset, tinnitus and decreased platelet adhesiveness. Other frequently used palliative anti-inflammatory therapeutic agents such as parenteral gold salts, penicillamine and corticosteroids such as prednisone also possess significant side effects. No treatment to date has had any consistent positive effect on the course of MS.
Recently, the art has described the use of specific deoxyribosides as anti-inflammatory agents. For instance, U.S. Pat. No. 4,481,197 to Rideout et al. relates to the use of unsubstituted 3-deaza-2'-deoxyadenosine derivatives in the treatment of inflammation. U.S. Pat. No. 4,381,344 to Rideout et al. relates to a process for the synthesis of deoxyribosides that utilizes a bacterial phosphorylase.
A deoxyriboside derivative, 2-chloro-2'-deoxyadenosine (CdA), has been found to be an effective agent for the treatment of chronic lymphocytic leukemia and some T cell malignancies. Carson et al. (1984) Proc. Natl. Acad. Sci. U.S.A., 81:2232-2236; Piro et al. (1988), Blood 72:1069-1073. Chronic lymphocytic leukemia is a malignancy of B lymphocytes that bear the Leu-l surface antigen.
The Leu-l B cells represent a minor proportion of the normal pool of B lymphocytes, usually less than 20 percent. The Leu-l B cells express surface markers that are typically found on monocytes (Mac-I antigen) and T-lymphocytes (Leu-l antigen). Approximately 10 percent of patients with chronic lymphocytic leukemia exhibit accompanying autoimmunity, and recently, Leu-l B cells have been implicated in the pathogenesis of autoimmune diseases.
Phase 1 studies on humans showed that infusion of increasing doses of 2-chloro-2'-deoxyadenosine [0.1-0.5 milligrams per kilogram of body weight per day (mg/kg/day)] yielded increasing plasma concentrations of the drug [10-50 nanomolar (nM)]. Those infusions indicated that the drug was well tolerated and did not induce nausea, vomiting or fever. The dose-limiting toxicity was bone marrow suppression, which usually occurred at doses greater than about 0.2 mg/kg/day or at plasma levels of greater than about 20 nM.
Other studies, Montgomery et al. (1959) J. Am. Chem, Soc., 82:463-468, indicated that 2-fluoroadenosine exhibits a relatively high degree of cytotoxicity. Those workers reported that C57 black mice implanted with Adenocarcinoma 755 (Ad755) could tolerate only about 1 milligram per kilogram of body weight. 2-Fluoroadenosine was found to be inactive at that level against Ad755 as well as leukemia L1210 and the Erlich ascites tumor.
U.S. Pat. No. 4,751,221 and its division No. 4,918,179 to Watanabe et al. describe the synthesis and use of several 2-substituted-2'-deoxy-2'-fluoroarabinofuranosyl nucleosides including adenine derivatives. Those compounds were said to have anti-tumor and antitrypanosomal biological activities. Cytotoxicity data showing anti-tumor activity of 2-amino-6-thiopurine, guanine and thiopurine derivatives against murine and human cell lines were reported.
U.S. Pat. No. 5,034,518 to Montgomery et al. teaches the synthesis of 2-substituted-2'-deoxy-2'-fluoroaraadenosines. Those compounds were said to have anticancer activity, and data for prolongation of life of mice transplanted with P388 leukemia cells were provided.
Chemotherapeutic agents are described hereinafter that exhibit substantial activity toward resting lymphocytes and monocytes. These agents are also useful in the treatment of autoimmune disorders.